Analytical device with variable angle of incidence

ABSTRACT

Apparatus for the determination of a chemical or biochemical species comprises a resonant optical biosensor (1-4) disposed in a light path between a pivotally-mounted source (5) of monochromatic light and a stationary detector (12) adapted to monitor some characteristic of the light. There is provided means (7) for causing pivotal motion of the light source (5) so as to vary the angle of incidence of the light on the sensor (1-4). Also provided is means for monitoring the instantaneous angle of incidence. The means for varying the angle of incidence of the light on the sensor may be a cam arrangement (7) acting on a pivoting member (6) carrying the light source (5), and the means for monitoring the instantaneous angle of incidence of the light on the sensor (1-4) may comprise means for monitoring the number of steps performed by a stepper motor driving the cam arrangement (7).

This invention relates to sensors, especially those termed biosensors,ie devices for the analysis of biologically active species such asantigens and antibodies in samples of biological origin. In particular,the invention relates to biosensors based on resonant optical phenomena,eg surface plasmon resonance or resonant attenuated or frustrated totalinternal reflection.

Many devices for the automatic determination of biochemical analytes insolution have been proposed in recent years. Typically, such devices(biosensors) include a sensitised coating layer which is located in theevanescent region of a resonant field. Detection of the analytetypically utilizes optical techniques such as, for example, surfaceplasmon resonance (SPR), and is based on changes in the thickness and/orrefractive index of the coating layer resulting from interaction of thatlayer with the analyte. This causes a change, eg in the angular positionof the resonance.

Other optical biosensors include a waveguide in which a beam of light ispropagated. The optical characteristics of the device are influenced bychanges occurring at the surface of the waveguide. One form of opticalbiosensor is based on frustrated total reflection. The principles offrustrated total reflection (FTR) are well-known; the technique isdescribed, for example, by Bosacchi and Oehrle [Applied Optics (1982),21, 2167-2173]. An FTR device for use in immunoassay is disclosed inEuropean Patent Application No 0205236A and comprises a cavity layerbounded on one side by the sample under investigation and on the otherside by a spacer layer which in turn is mounted on a substrate. Thesubstrate-spacer layer interface is irradiated with monochromaticradiation such that total reflection occurs, the associated evanescentfield penetrating through the spacer layer. If the thickness of thespacer layer is correct and the incident parallel wave vector matchesone of the resonant mode propagation constants, the total reflection isfrustrated and radiation is coupled into the cavity layer. The cavitylayer must be composed of material which has a higher refractive indexthan the spacer layer and which is transparent at the wavelength of theincident radiation.

In devices of this kind, the position of resonance is monitored byvarying the angle at which light is incident on the sensor. The scanningof angle may be performed either sequentially or simultaneously ie byvarying the angle of incidence of a parallel beam of light or bysimultaneously irradiating over a range of angles using a fan-shapedbeam of light as described (in connection with SPR) in European PatentApplication No 0305109A. In the former case, prior proposals haveinvolved a single-channel detector which is mechanically scanned over arange of angles; this necessitates synchronisation of the movement ofthe light source and the detector. In the latter case, in which a rangeof angles is irradiated simultaneously, it is generally necessary to usea multi-channel detector having angular resolution. This leads torelatively high manufacturing costs.

There has now been devised an apparatus involving the use of a resonantoptical sensor for the determination of a chemical or biochemicalspecies, which overcomes or substantially mitigates some or all of thedisadvantages of the prior art arrangements described above.

According to the invention, there is provided apparatus for thedetermination of a chemical or biochemical species, comprising aresonant optical biosensor disposed in a light path between apivotally-mounted source of monochromatic light and a stationarydetector adapted to monitor some characteristic of the light, therebeing provided means for causing pivotal motion of the light source soas to vary the angle of incidence of the light on the sensor and meansfor monitoring the instantaneous angle of incidence.

The apparatus according to the invention is advantageous primarily inthat it is of relatively simple construction and uses only asingle-channel detector. Also, the means for monitoring theinstantaneous angle of incidence provides an accurate correlation of theoutput characteristics of the light beam with that angle.

Any convenient source of monochromatic light may be used. The choice ofsource will depend inter alia on the particular form of sensor used. Inthis context, `light` may include not only visible light but alsowavelengths above and below this range, eg in the ultra-violet andinfra-red.

The means for varying the angle of incidence of the light on the sensormay be mechanical, eg a stepper motor-driven cam arrangement acting on apivoting member carrying the light source and associated optics. Theangle of incidence is preferably varied over only that range of anglesin which resonance occurs.

The means for monitoring the instantaneous angle of incidence of thelight on the sensor may comprise means for monitoring the number ofsteps performed by a stepper motor driving the cam arrangement, therelationship between the cam position and the angle of incidence beingknown. A non-contact zero position indicator may be used on the camarrangement to ensure that the stepper motor is performing as expected.

The characteristic of the light which is monitored may be anycharacteristic which changes at resonance, eg the phase of reflectedradiation or, in some cases, the intensity.

The sensor is preferably an FTR sensor. Such a sensor will generallyinclude an optical structure comprising

a) a cavity layer of transparent dielectric material of refractive indexn₃,

b) a dielectric substrate of refractive index n₁, and

c) interposed between the cavity layer and the substrate, a dielectricspacer layer of refractive index n₂.

In use, the interface between the substrate and the spacer layer isirradiated with light such that internal reflection occurs. Resonantpropagation of a guided mode in the cavity layer will occur, for a givenwavelength, at a particular angle of incidence of the excitingradiation.

The angular position of the resonant effect depends on variousparameters of the sensor device, such as the refractive indices andthicknesses of the various layers. In general, it is a pre-requisitethat the refractive index n₃ of the cavity layer and the refractiveindex n₁ of the substrate should both exceed the refractive index n₂ ofthe spacer layer. Also, since at least one mode must exist in the cavityto achieve resonance, the cavity layer must exceed a certain minimumthickness.

The cavity layer is preferably a thin-film of dielectric material.Suitable materials for the cavity layer include zirconium dioxide,titanium dioxide, aluminium oxide and tantalum oxide.

The cavity layer may be prepared by known techniques, eg vacuumevaporation, sputtering, chemical vapour deposition or in-diffusion.

The dielectric spacer layer must have a lower refractive index than boththe cavity layer and the substrate. The layer may, for example, comprisean evaporated or sputtered layer of magnesium fluoride. In this case aninfra-red light injection laser may be used as light source. The lightfrom such a source typically has a wavelength around 800 nm. Othersuitable materials include lithium fluoride and silicon dioxide. Apartfrom the evaporation and sputtering techniques mentioned above, thespacer layer may be deposited on the substrate by a sol-gel process, orbe formed by chemical reaction with the substrate.

The sol-gel process is particularly preferred where the spacer layer isof silicon dioxide.

The refractive index of the substrate (n₁) must be greater than that(n₂) of the spacer layer but the thickness of the substrate is generallynot critical.

By contrast, the thickness of the cavity layer must be so chosen thatresonance occurs within an appropriate range of coupling angles. Thespacer layer will typically have a thickness of the order of severalhundred nanometres, say from about 200 nm to 2000 nm, more preferably500 to 1500 nm, eg 1000 nm. The cavity layer typically has a thicknessof a few tens of nanometres, say 10 to 200 nm, more preferably 30 to 150nm, eg 100 nm.

It is particularly preferred that the cavity layer has a thickness of 30to 150 nm and comprises a material selected from zirconium dioxide,titanium dioxide, tantalum oxide and aluminium oxide, and the spacerlayer has a thickness of 500 to 1500 nm and comprises a materialselected from magnesium fluoride, lithium fluoride and silicon dioxide,the choice of materials being such that the refractive index of thespacer layer is less than that of the cavity layer.

Preferred materials for the cavity layer and the spacer layer aretantalum oxide and silicon dioxide respectively.

At resonance, the incident light is coupled into the cavity layer byFTR, propagates a certain distance along the cavity layer, and couplesback out (also by FTR). The propagation distance depends on the variousdevice parameters but is typically of the order of 1 or 2 mm.

At resonance the reflected light will undergo a phase change, and it isthis which may be detected. Alternatively, as described in ourco-pending International Patent Application No PCT/GB91/01161 the cavitylayer and/or spacer layer may absorb at resonance, resulting in areduction in the intensity of the reflected light.

For use in the determination of biochemical species, the surface of thesensor, ie the surface of the cavity layer in the case of an FTR sensor,will generally be sensitised by having biomolecules, eg specific bindingpartners for the analyte(s) under test, immobilised upon it. Theimmobilised biochemicals may be covalently bound to the sensor surfaceby methods which are well known to those skilled in the art.

The invention will now be described in more detail, by way ofillustration only, with reference to the accompanying drawings in which

FIG. 1 is a schematic view (not to scale) of an apparatus according tothe invention,

FIG. 2 depicts the dependence of the intensity of the detected light onthe angle of incidence, and

FIG. 3 is a plan view of part of a second embodiment of an apparatusaccording to the invention in which two regions of a sensor areirradiated simultaneously.

Referring first to FIG. 1, a biosensor comprises a glass prism 1 coatedover an area of its base with a first coating 2 of magnesium fluorideand a second coating 3 of titanium dioxide. The prism 1 and first andsecond coatings 2,3 together constitute a resonant optical structure,the first coating 2 acting as a spacer layer and the second coating 3 asa cavity layer. The first coating 2 has a thickness of approximately1000 nm and the second coating 3 a thickness of approximately 100 nm.

Immobilised on the surface of the second coating 3 is a layer 4 ofimmobilised biochemicals, which act as specific binding partner for theanalyte under test.

The interface between the base of the prism 1 and the first coating 2 isirradiated by a beam of monochromatic light from a laser 5 which ismounted on a pivoted arm 6. The arm 6 can be moved through a range ofangles by means of a cam 7 which is driven by a stepper motor (notshown).

Also mounted on the arm 6, between the laser 5 and the prism 1, arecollimating optics 8 and a polariser 9. The polariser 9 is arranged toproduce linearly polarised light with two components: transverseelectric (TE) and transverse magnetic (TM). The polariser is set at 45°to the TE and TM transmission axes and thus provides equal components ofTE and TM light.

All the light incident on the interface between the base of the prism 1and the first coating 2 is reflected, resonance being detected as achange of phase of the reflected light.

The reflected light is passed through a compensator 12 to a polarisationanalyser 13. The compensator 12, which may be of any conventional form,is manually adjusted to remove any phase difference which is introducedinto the TE and TM components on reflection and by birefringence in theoptical path.

The analyser 13 is arranged at 90° to the polariser 9. The TE and TMcomponents are interfered at the analyser to allow the phase change tobe detected. Off resonance both components undergo a similar phase shifton reflection and the relative phase between the components is adjustedby the compensator 12 to give zero transmission through the analyser 13.This will apply for all angles except near resonance. Near resonance ofeither component, the phase shift between the TE and TM components willvary rapidly with angle, resulting in maximum throughput of lightthrough the analyser 13 at resonance.

Light passing through the analyser 13 is focussed by a cylindricalcondenser lens arrangement 14 onto a detector 15. The condenser lensarrangement 14 is located so as to collect light from all incidentangles onto the detector 15. This minimises the effects of positioningerrors.

In use, the angle of incidence of light on the interface between thebase of the prism 1 and the first coating layer 2 is varied by rotationof the cam 7. The incident light beam is therefore scanned through arange of incident angles including the resonant angle. Off-resonance nolight intensity is detected at the detector 15; as resonance isapproached, the detected light intensity increases and then falls. Theincrease in intensity is correlated with the angle of incidence,enabling the angular position of the resonance to be determined. Theinstantaneous angle of incidence is determined from the instantaneousposition of the cam 7, the relationship between the cam position and theangle of incidence being known.

When the layer of immobilised biochemicals 4 is contacted with a samplecontaining the analyte under test, specific binding occurs between thebiochemicals and the analyte molecules, resulting in a change in therefractive index in the vicinity of the surface of the device. This inturn results in a shift in the position of the resonance. FIG. 2 shows aplot of the measured signal intensity against angle of incidence beforeand (dotted line) after complexation of the immobilised biochemicalswith the analyte.

In the embodiment shown in FIG. 3, there are two separate patches 31,32of immobilised biochemicals on the surface of the prism 33. Each patch31,32 is irradiated with a separate beam of incident radiation, theangle of incidence being scanned as described above.

Each reflected beam is passed through a compensator 34 and apolarisation analyser 35, and then focussed by a cylindrical condenserlens arrangement 36 onto two detectors 37,38. Again, the lensarrangement 36 is located so as to collect light from all incidentangles onto the corresponding detector 37,38. The cylindrical condenserlens arrangement 36 has power only in one dimension, thereby preservingthe spatial separation of the light beams reflected from the separatepatches 31,32.

As shown in FIG. 3, reflecting mirrors 39 are placed in the beam toenable the detectors 37,38 to be spatially separated.

We claim:
 1. Apparatus for the determination of a chemical orbiochemical species, said apparatus comprising a pivotally-mountedsource of monochromatic light, a stationary detector adapted to monitora characteristic of the light, a resonant optical biosensor disposed ina light path between said source and said detector, means for causingpivotal motion of said source so as to vary the angle of incidence ofthe light on said sensor, and means for monitoring the instantaneousangle of incidence of the light on said sensor, wherein said resonantoptical biosensor is a frustrated total reflection sensor comprising:acavity layer of transparent dielectric material of refractive index n₃,a dielectric substrate of refractive index n₁, and interposed betweensaid cavity layer and said substrate, a dielectric spacer layer ofrefractive index n₂, wherein n₂ is less than n₁ and n₂ is less than n₃.2. Apparatus as claimed in claim 1, wherein the angle of incidence isvariable only over that range of angles in which resonance occurs. 3.Apparatus as claimed in claim 1, wherein said means for causing pivotalmotion of said source comprises a cam arrangement acting on a pivotingmember carrying said source.
 4. Apparatus as claimed in claim 3, whereinsaid means for monitoring the instantaneous angle of incidence of thelight on said sensor comprises means for monitoring the number of stepsperformed by a stepper motor driving said cam arrangement.
 5. Apparatusas claimed in claim 3, further comprising a non-contact zero positionindicator for said cam arrangement.